title: An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva.
abstract: Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.
authors.affiliation: Faculty of Dental Medicine, Institute of Dental Sciences, The Hebrew University, Jerusalem, Israel.
authors.forename: Omer
authors.initials: O
authors.lastname: Deutsch
authors.forename: Yoram
authors.initials: Y
authors.lastname: Fleissig
authors.forename: Batia
authors.initials: B
authors.lastname: Zaks
authors.forename: Guy
authors.initials: G
authors.lastname: Krief
authors.forename: Doron J
authors.initials: DJ
authors.lastname: Aframian
authors.forename: Aaron
authors.initials: A
authors.lastname: Palmon
mesh_terms: D000327:Adsorption; D015415:Biomarkers / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012463:Saliva / Q000737:chemistry*; D013213:Starch / Q000737:chemistry; D000516:alpha-Amylases / Q000302:isolation & purification*
chemical_list: D015415:Biomarkers; D020543:Proteome; D013213:Starch; D000516:alpha-Amylases